Potency Assessment of Dendritic Cell Anticancer Vaccine: Validation of the Co-Flow DC Assay

Abstract

For many years, oncological clinical trials have taken advantage of dendritic cells (DC) for the design of DC-based cellular therapies. This has required the design of suitable quality control assays to evaluate the potency of these products. The purpose of our work was to develop and validate a novel bioassay that uses flow cytometry as a read-out measurement. In this method, CD3+ cells are labeled with a fluorescent dye and the DC costimulatory activity is measured by the degree of T cell proliferation caused by the DC–T cell interaction. The validation of the method was achieved by the evaluation of essential analytical parameters defined by international guidelines. Our results demonstrated that the method could be considered specific, selective, and robust. The comparison between measured values and estimated true values confirmed a high level of accuracy and a lack of systematic error. Repeated experiments have shown the reproducibility of the assay and the proportionality between the potency and the DC amount has proven its linearity. Our results suggest that the method is compliant with the guidelines and could be adopted as a quality control assay or batch-release testing within GMP facilities.

Stability Program in Dendritic Cell Vaccines: A “Real-World” Experience in the Immuno-Gene Therapy Factory of Romagna Cancer Center

“…The variability of the potency data among different batches was probably linked to the complexity of the method or to the intrinsic biological activity of cellular product; thus, method standardization or eventual replacement with other validated tests, such as the Co-Flow DC [ 14 ], should be considered. This cytofluorimetric method could allow the evaluation of the absolute number of proliferating T cells in co-culture with mDCs and a suboptimal amount of anti-CD3 antibody and the accurate live cell seeding using Annexin V assay.…”

“…However, this in vitro assay does not allow to separate the degree of stimulation due to the presentation of alloantigens from that induced by the costimulatory activity of DCs. Alternatively, DCs’ costimulatory activity could be indirectly assessed by the expression analysis of phenotypic markers [ 11 ] or using other assays such as the COSTIM and Co-Flow DC assay [ 12 , 13 , 14 ].…”

Potency assays and biomarkers for cell-based advanced therapy medicinal products

“…Furthermore, such assays, which are based on proliferation measured over several days and employ different donors, show intrinsic variability. Specifications also vary between laboratories and are based on the validation data (31,93,94,104,105) (Table 3). Thus, no standardized assays are defined for DCs.…”

“…Potency assays may in some cases be used as a GMP release assay but are more likely employed in the validation phase and/or used for information only in the context of clinical trials. They may be performed before cryopreservation or on cryopreserved cells (31,93,94,104,105). Immunophenotyping to define the identity of the clinical grade DC products is therefore often used as surrogate for potency assays (positivity for CD11c, CD80/83/86, HLA-DR, CD209 and negativity for CD14, CD3 CD19), but the set of markers used and the specifications somewhat vary between laboratories (31,93,94,104,105).…”